Blood clotting begins when platelets adhere at a lesion site in the cut wall of an injured blood vessel. In a cascade of enzymatically regulated reactions, soluble fibrinogen molecules are converted by the enzyme thrombin to insoluble strands of fibrin that hold the platelets together in a thrombus. At each step in the cascade, a protein precursor is converted to a protease that cleaves the next protein precursor in the series. Co-factors are required at most of the steps.
The human factor VIII (FVIII) is a plasma glycoprotein that acts as a cofactor for the serine protease factor FIXa to activate FX in the intrinsic cascade of blood coagulation. Factor VIII circulates as an inactive precursor at a very low concentration in blood, bound tightly and non-covalently to von Willebrand factor. Factor VIII is proteolytically activated by thrombin or factor Xa, which dissociates it from von Willebrand factor and activates its procoagulant function in the cascade (Hoyer, 1981; Kane and Davie, 1988). In its active form, the protein FVIIIa is a cofactor that increases the catalytic efficiency of factor IXa toward factor X activation by several orders of magnitude.
Quantitative or qualitative deficiency in FVIII results in a bleeding disorder called hemophilia A (Scandella et al., 1998; Rick et al., 2003). Severe hemophiliacs number about 17,000 in the United States. These patients can suffer uncontrolled internal bleeding that may result in serious symptoms ranging from inflammatory reactions in joints to early death. These patients can be treated with human FVIII, which will restore the blood's normal clotting ability if administered with sufficient frequency and concentration. Hemophiliacs require daily replacement of factor VIII to prevent bleeding and the resulting deforming hemophilic arthropathy.
Several commercial preparations of human plasma-derived FVIII of varying degrees of purity are available for the treatment of hemophilia A. These products are derived from blood plasma of human donors treated to remove viruses, or prepared by recombinant means from cultures of cells that carry genetically engineered recombinant (r) full-length or truncated FVIII (Brackmann et al., 1993; Lusher et al., 1993); Bihoreau et al., 1991; Pipe and Kaufman, 1997; Sandberg et al., 2001).
Unfortunately, human factor VIII is unstable at physiologic concentrations and pH, and is present in blood at an extremely low concentration. Problems in therapeutic use occur due to difficulty in isolation and purification, immunogenicity, and the necessity of removing the AIDS and hepatitis infectivity risk.
Accurate assessment of the quantity and quality of FVIII is critical to successful outcome in hemophilia patients undergoing FVIII replacement therapy. Current assays of quantification of FVIII products and concentrates involve bioassays including clotting assays and generation of FXa (Langdell et al., 1953; Niemetz and Nossel, 1969; Over, 1986; Kemball-Cook et al., 1993). Thus currently available assays measure FVIII concentration only indirectly (Hoyer, 1981; Kane and Davie, 1988; Chavin and Fay, 1989; Foster and Zimmerman, 1989; Fay, 1993; Lenting et al., 1998). Unfortunately such indirect assays exhibit problems of poor reproducibility and lack of precision due to complex reaction kinetics.
A particular problem associated with FVIII preparations is the presence of FVIII degradation product. This is undesirable for several reasons. First, much more FVIII is required to achieve a desired therapeutic goal. Also, degradation products can interfere with FVIII function by interacting with substrate proteins, reducing the efficiency of FVIII activation by the substrates. Use of excess FVIII in patients is also undesirable as it can enhance production of neutralizing FVIII-specific antibodies (Scandella et al., 1998; El Alfy et al., 2000; Klinge et al., 2001; Lindgren et al., 2002). Available bioassays for FVIII reflect only the concentration of fully functional FVIII. Thus, if FVIII preparations contain degraded or inactive FVIII, such products are not detectable by bioassays.
To enhance ability to precisely assess the quality of FVIII preparations in a timely and cost effective manner, there exists a clear need for sensitive assays that can determine both the concentration and the biological activity of FVIII in FVIII preparations.
As discussed, FVIII is present in the blood of normal subjects at a very low concentration (about 100-700 pM). In severe hemophilia patients, FVIII concentrations are below 1% physiological concentration. Such low FVIII concentrations are below the level of detection of existing assay methods. A great improvement in the diagnosis and management of hemophilia patients would be achieved if it were possible to accurately measure FVIII levels in the plasma of these patients. Thus both for monitoring FVIII levels in patients suspected of having a blood clotting disorder, and in severe, moderate, and mild hemophilia patients undergoing FVIII replacement therapy, there is an unmet need for highly specific and sensitive assays that can detect FVIII at physiological concentrations and significantly below.